Multianalyte Molecular Analysis Using Application-Specific Random Particle Arrays

ABSTRACT

The present invention provides methods and apparatus for the application of a particle array in bioassay format to perform qualitative and/or quantitative molecular interaction analysis between two classes of molecules (an analyte and a binding agent). The methods and apparatus disclosed herein permit the determination of the presence or absence of association, the strength of association, and/or the rate of association and dissociation governing the binding interactions between the binding agents and the analyte molecules. The present invention is especially useful for performing multiplexed (parallel) assays for qualitative and/or quantitative analysis of binding interactions of a number of analyte molecules in a sample.

FIELD OF THE INVENTION

The present invention relates to mutiplexed bioassays for analyzingbinding interactions between analytes and binding agents, includingmethods for determining the affinity constants and kinetic propertiesassociated with analyte-binding agent interactions.

BACKGROUND OF THE INVENTION

The imprinting of multiple binding agents such as antibodies andoligonucleotides on planar substrates in the form of spots or stripesfacilitates the simultaneous monitoring of multiple analytes such asantigens and DNA in parallel (“multiplexed”) binding assays. Theminiaturization of this array format for increasing assay throughput andstudying binding kinetics are described, for example, in R. Ekins, F. W.Chu, Clin. Chem. 37, 955-967 (1991); E. M. Southern, U. Maskos, J. K.Elder, Genomics 13, 1008-1017 (1992). In recent years, this approach hasattracted substantial interest particularly in connection withperforming extensive genetic analysis, as illustrated in G. Ramsay, Nat.Biotechnol. 16, 40-44 (1998); P. Brown, D. Botstein, Nat. Genet. 21,33-37 (1999); D. Duggan, M. Bittner, Y. Chen, P. Meltzer, J. M. Trent,Nat. Genet. 21, 10-14 (1999); R. Lipshutz, S. P. A. Fodor, T. KGingeras, D. J. Lockhart, Nat. Genet. 21, 20-24 (1999).

The principal techniques of array fabrication reported to date include:refinements of the original “spotting” in the form of pin transfer orink jet printing of small aliquots of probe solution onto varioussubstrates, as illustrated in V. G. Cheung, et al., Nat. Genet. 21,15-19 (1999); sequential electrophoretic deposition of binding agents inindividually electrically addressable substrate regions, as illustratedin J. Cheng, et al., Nat. Biotechnol. 541-546 (1998); and methodsfacilitating spatially resolved in-situ synthesis of oligonucleotides,as illustrated in U. Maskos, E. M. Southern, Nucleic Acids Res. 20,1679-1684 (1992); S. P. A. Fodor, et al., Science 251, 767-773 (1991) orcopolymerization of oligonucleotides, as illustrated in A. V.Vasiliskov, et al., BioTechniques 27, 592-606 (1999). These techniquesproduce spatially encoded arrays in which the position within the arrayindicates the chemical identity of any constituent probe.

The reproducible fabrication of customized arrays by these techniquesrequires the control of microfluidics and/or photochemical manipulationsof considerable complexity to ensure consistent performance inquantitative assays. Microfluidic spotting to produce, in quantitativelyreproducible fashion, deposited features of 100 μm diameter involvesdispensing of nanoliter aliquots with tight volume control, a task thatexceeds the capabilities of currently available fluid handlingmethodologies. In addition, exposure of binding agents to air during thedeposition process, typically several hours' in duration, hasuncontrollable impact on the molecular configuration and theaccessibility of the binding agents in subsequent binding assays.In-situ array synthesis relies on a sequence of multiple masking andphotochemical reaction steps which must be redesigned to accommodate anychanges in array composition. Finally, assay performance must beassessed “in-situ” for each array subsequent to immobilization ofbinding agents, an aspect of array manufacturing which raises difficultquality control and implementation issues.

SUMMARY OF THE INVENTION

The present invention provides methods and apparatus for the applicationof a particle array in bioassay format to perform qualitative and/orquantitative molecular interaction analysis between two classes ofmolecules (an analyte and a binding agent). The methods and apparatusdisclosed herein permit the determination of the presence or absence ofassociation, the strength of association, and/or the rate of associationand dissociation governing the binding interactions between the bindingagents and the analyte molecules. The present invention is especiallyuseful for performing multiplexed (parallel) assays for qualitativeand/or quantitative analysis of binding interactions.

The terms “analyte” and “binding agent” refer to molecules involved inbinding interactions. In one example, analyte and binding agent includeDNA or RNA fragments (e.g., oligonucleotide), and binding of thosefragments to their complementary sequences (hybridization) is analyzed.In another example, binding interactions between ligands and receptorsare analyzed. Examples of analytes and binding agents also includeaptamers, peptides and proteins (e.g., antibodies), antigens, and smallorganic molecules.

The term “particles” refer to colloidal particles, including beadedpolymer resins (“beads”).

The present invention also provides automated, on-demand fabrication ofplanar arrays composed of a selected mixture of chemically distinctbeads (e.g., encoded beads) which are disposed on a substrate surface inaccordance with a selected spatial configuration, as described above. Inthis approach, the beads are functionized to display binding agents. Forexample, the binding agents may be attached to the beads, preferably bycovalent bond. The subsequent quality control and performance evaluationare conducted off-line and are independent from the process of arrayassembly. The separation of steps such as bead encoding,functionalization and testing; substrate design, processing andevaluation; custom assembly of application-specific arrays; and on-linedecoding of arrays enable an otherwise elusive combination offlexibility, reliability and low cost by permitting systematic processcontrol.

The methods disclosed herein permit rapid customization of DNA orprotein arrays without the need for process redesign and avoid problemscontributing to spot-to-spot as well as chip-to-chip variability.Furthermore, the bead array format permits chip-independentcharacterization of beads as well as optimization of assay conditions.In addition, multiple bead arrays can be formed simultaneously indiscrete fluid compartments maintained on the same chip, permitting theconcurrent processing of multiple samples.

BRIEF DESCRIPTION OF DRAWINGS

Other objects, features and advantages of the invention discussed in theabove brief explanation will be more clearly understood when takentogether with the following detailed description of an embodiment whichwill be understood as being illustrative only, and the accompanyingdrawings reflecting aspects of that embodiment, in which:

FIG. 1 is an illustration of process flow including the production ofrandom encoded bead arrays and their use in multiplexed assays

FIG. 2 is an Illustration of the functionalization of beads

FIG. 3 is an illustration of steps in chip design and wafer-scaleproduction

FIG. 4 is an illustration of on-demand assembly of random encoded arrays

FIG. 5 is an illustration of palmtop microlab

FIG. 6 is a schematic illustration of assay and decoding images used inREAD process

FIG. 7 is a flow chart summarizing algorithms and steps in the analysisof images

FIG. 8 is an illustration of steps in the decomposition of assay imagesaccording to bead type by application of the image analysis algorithmsummarized in FIG. 7.

FIG. 9 is an illustration of optically programmable array assembly ofrandom encoded arrays

FIG. 10 is an illustration of an array composed of random encodedsubarrays

FIG. 11 is an illustration of stations in an automated chip-scale beadarray manufacturing and QC process

FIG. 12 is an illustration of quantitative binding curves for twocytokines

FIG. 13 is an illustration of array design for polymorphism analysis

FIG. 14 is a fluorescence micrograph of assay and decoding imagesrecorded from one subarray shown in FIG. 13 in the course ofpolymorphism analysis

FIG. 15 is an illustration of assay results in the form of intensityhistograms obtained from the analysis of assay images such as the oneillustrated in FIG. 14.

FIG. 16 is an illustration of design of a “looped probe” forhybridization assays

FIGS. 17A and 17B are fluorescence micrographs of assay and decodingimages recorded in the course of the analysis of multiple cytokines

FIGS. 18A and 18B are illustrations of numerical simulations ofcross-correlations in receptor-ligand systems with multiple competingreceptor-ligand interactions

FIG. 19 is an illustration of numerical simulations of receptor-ligandassociation and disassociation kinetics

FIG. 20 is an illustration of integrated sample capture using magneticcapture beads and array-based detection using READ

FIG. 21 is an illustration of multi-step assays using encoded magneticbeads to integrate gene-specific capture, on-bead reverse transcriptionand post-assay array assembly

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The fabrication of application-specific bead arrays may involve multipleprocesses in a multi-step sequence which may be automated using existingliquid handling technology and laboratory automation. The process ofRandom Encoded Array Detection (READ) includes the fabrication of custombead arrays as well as the use of such arrays in bioassays, includingassays involving multiplexed molecular interaction analysis of analyteand binding agent molecules, including DNA and protein analysis.

FIG. 1 provides a schematic overview of the functional components andprocess flow by which custom bead arrays may be prepared and used inperforming multiplexed biomolecular analysis according to the presentinvention. The array is prepared by employing separate batch processesto produce application-specific substrates (e.g., chip at the waferscale) and to produce beads that are chemically encoded and biologicallyfunctionalized (e.g., at the scale of ˜10̂8 beads/100 μl of suspension).The beads subjected to respective quality control (QC) steps prior toarray assembly, such as the determination of morphological andelectrical characteristics. In addition, actual assays are performed onbeads in suspension, before they are introduced to the substrate, tooptimize assay conditions, generally with the objective to maximizeassay sensitivity and specificity and to minimize bead-to-beadvariations. For substrates, QC steps may include optical inspection,ellipsometry and electrical transport measurements.

Once the chemically encoded and biologically functionalized beads arecombined with the substrate (e.g., chip), the Light-controlledElectrokinetic Assembly of Particles near Surfaces (LEAPS) may be usedfor rapid assembly of dense arrays on a designated area on the substratewithin the same fluidic phase, avoiding problems contributing tospot-to-spot as well as chip-to-chip variability without the need forretooling or process redesign. Furthermore, the bead array formatpermits chip-independent characterization of beads as well asoptimization of assay conditions. In addition, multiple bead arrays canbe formed simultaneously in discrete fluid compartments maintained onthe same chip. Once formed, these multiple bead arrays may be used forconcurrent processing of multiple samples. The integration of LEAPS withmicrofluidics produces a self-contained, miniaturized, opticallyprogrammable platform for parallel protein and DNA analysis. LEAPSrefers to methods of moving particles suspended in the interface betweenan electrolyte solution and an electrode and is described in U.S. patentapplication Ser. No. 09/171,550 (also PCT International Application No.PCT/US97/08159) entitled Light-controlled Electrokinetic Assembly ofParticles near Surfaces, which is incorporated herein by reference inits entirety. Also incorporated herein by reference in its entirety isU.S. patent application Ser. No. 09/397,793 (also PCT InternationalApplication PCT/US00/25466), entitled “System and Method forProgrammable Pattern Generation.

In certain embodiments of the present invention, chemical encoding maybe accomplished by staining beads with sets of optically distinguishabletags, such as those containing one or more fluorophore dyes spectrallydistinguishable by excitation wavelength, emission wavelength,excited-state lifetime or emission intensity. The opticallydistinguishable tags made be used to stain beads in specified ratios, asdisclosed, for example, in Fulwyler, U.S. Pat. No. 4,717,655 (Jan. 5,1988). Staining may also be accomplished by swelling of particles inaccordance with methods known to those skilled in the art, [Molday,Dreyer, Rembaum & Yen, J. Mol Biol 64, 75-88 (1975); L. Bangs, “Uniformlatex Particles, Seragen Diagnostics, 1984]. For example, up to twelvetypes of beads were encoded by swelling and bulk staining with twocolors, each individually in four intensity levels, and mixed in fournominal molar ratios. Combinatorial color codes for exterior andinterior surfaces is disclosed in International Application No. PCT/US98/10719, which is incorporated herein by reference in its entirety.

Beads are functionalized by binding agent molecules attached thereto,the molecule including DNA (oligonucleotides) or RNA fragments, peptidesor proteins, aptamers and small organic molecules in accordanceprocesses known in the art, e.g., with one of several coupling reactionsof the known art (G. T. Hermanson, Bioconjugate Techniques (AcademicPress, 1996); L. Illum, P. D. E. Jones, Methods in Enzymology 112, 67-84(1985). In certain embodiments of the invention, the functionalizedbeads have binding agent molecules (e.g., DNA, RNA or protein)covalently bonded to the beads. Beads may be stored in a buffered bulksuspension until needed. Functionalization typically requires one-stepor two-step reactions which may be performed in parallel using standardliquid handling robotics and a 96-well format to covalently attach anyof a number of desirable functionalities to designated beads, asillustrated in FIG. 2. In a preferred embodiment, beads of core-shellarchitecture will be used, the shell composed in the form of a thinpolymeric blocking layer whose preferred composition is selected; andfunctionalization performed in accordance with the targeted assayapplication, as known in the art. Samples may be drawn for automated QCmeasurements. Each batch of beads provides material for hundreds ofthousands of chips so that chip-to-chip variations are minimized.

Substrates (e.g., chips) used in the present invention may be patternedin accordance with the interfacial patterning methods of LEAPS by, e.g.,patterned growth of oxide or other dielectric materials to create adesired configuration of impedance gradients in the presence of anapplied AC electric field. Patterns may be designed so as to produce adesired configuration of AC field-induced fluid flow and correspondingparticle transport. Substrates may be patterned on a wafer scale byinvoking semiconductor processing technology, as illustrated in FIG. 3.In addition, substrates may be compartmentalized by depositing a thinfilm of a UV-patternable, optically transparent polymer to affix to thesubstrate a desired layout of fluidic conduits and compartments toconfine fluid in one or several discrete compartments, therebyaccommodating multiple samples on a given substrate.

In certain embodiments of the invention, the bead array is prepared byproviding a first planar electrode that is in substantially parallel toa second planar electrode (“sandwich” configuration) with the twoelectrodes being separated by a gap and containing an electrolytesolution. The surface or the interior of the second planar electrode ispatterned with the interfacial patterning method. Encoded andfunctionalized beads are introduced into the gap. When an AC voltage isapplied to the gap, the beads form a random encoded array on the secondelectrode (e.g., “chip”). And, also using LEAPS, an array of beads maybe formed on a light-sensitive electrode (“chip”). Preferably, thesandwich configuration described above is also used with a planar lightsensitive electrode and another planar electrode. Once again, the twoelectrodes are separated by the a gap and contain an electrolytesolution. The functionalized and encoded beads are introduced into thegap. Upon application of an AC voltage in combination with a light, thebeads form an array on the light-sensitive electrode.

In certain embodiments, the application-specific bead arrays useful inthe present invention may be produced by picking aliquots of designatedencoded beads from individual reservoirs in accordance with thespecified array composition and “pooled”; aliquots of pooled suspensionare dispensed onto selected substrate (e.g., chips) in a mannerpreventing the initial fusion of aliquots. Aliquots form a multiplicityof planar random subarrays of encoded beads, each subarray representingbeads drawn from a distinct pool and the physical array layout uniquelycorresponding to the identity of aliquots drawn from pooled beadpopulations.

Planar arrays or assemblies of encoded on a substrate which arechemically or physically encoded may be used. To this, spatial encodingmay also be added to increase the number of assays that may beconducted. Spatial encoding, for example, can be accomplished within asingle fluid phase in the course of array assembly by invokingLight-controlled Electrokinetic Assembly of Particles near Surfaces(LEAPS) to assemble planar bead arrays in any desired configuration inresponse to alternating electric fields and/or in accordance withpatterns of light projected onto the substrate. LEAPS creates lateralgradients in the impedance of the interface between silicon chip andsolution to modulate the electrohydrodynamic forces that mediate arrayassembly. Electrical requirements are modest: low AC voltages oftypically less than 10V_(pp) are applied across a fluid gap of typically100 μm between two planar electrodes. This assembly process is rapid andit is optically programmable: arrays containing thousands of beads areformed within seconds under electric field. The formation of multiplesubarrays, can also occur in multiple fluid phases maintained on acompartmentalized chip surface.

The multiplexed assays of the present invention may also be performedusing beads encoded beads that are assembled, but not in an array, onthe substrate surface. For example, by spotting bead suspensions intomultiple regions of the substrate and allowing beads to settle undergravity, assemblies of beads can be formed on the substrate. In contrastto the bead arrays formed by LEAPS, these assemblies generally assumelow-density, disorder configurations. However, the combination ofspatial and color encoding attained by spotting mixtures of chemicallyencoded beads into a multiplicity of discrete positions on the substratestill provides a degree of multiplexing that is sufficient for certainbiological assays.

Binding interaction between the binding agent on those beads and ananalyte may be performed either before or after the encoded array isassembled on the substrate. For example, the bead array may be formedafter the assay, subsequent to which an assay image and a decoding imagemay be taken of the array. Alternatively, the beads may be assembled inan array and immobilized by physical or chemical means to produce randomencoded arrays, e.g., with the appearance of the array shown in FIG. 10.The arrays may be immobilized, for example, by application of a DCvoltage to produce random encoded arrays with the appearance of thearray shown in FIG. 10. The DC voltage, set to typically 5-7 V (forbeads in the range of 2-6 μm and for a gap size of 100-150 μm) andapplied for <30s in “reverse bias” configuration so that an n-dopedsilicon substrate would form the anode, causes the array to becompressed to an extent facilitating contact between adjacent beadswithin the array and simultaneously causes beads to be moved toward theregion of high electric field in immediate proximity of the electrodesurface. Once in sufficiently close proximity, beads are anchored by vander Waals forces mediating physical adsorption. This adsorption processis facilitated by providing on the bead surface a population of“tethers” extending from the bead surface; polylysine and streptavidinhave been used for this purpose.

In certain embodiments, the particle arrays may be immobilized bychemical means, e.g, by forming a composite gel-particle film. In oneexemplary method for forming such gel-composite particle films, asuspension of microparticles is provided which also contain allingredients for subsequent in-situ gel formation, namely monomer,crosslinker and initiator. The particles are assembled into a planarassembly on a substrate by application of LEAPS, e.g., AC voltages of1-20 V_(p-p) in a frequency range from 100's of hertz to severalkilohertz are applied between the electrodes across the fluid gap.Following array assembly, and in the presence of the applied AC voltage,polymerization of the fluid phase is triggered by thermally heating thecell ˜40-45° C. using an IR lamp or photometrically using a mercury lampsource, to effectively entrap the particle array within a gel. Gels maybe composed of a mixture of acrylamide and bisacrylamide of varyingmonomer concentrations from 20% to 5% (acrylamide:bisacrylamide=37.5:1,molar ratio), or any other low viscosity water soluble monomer ormonomer mixture may be used as well. Chemically immobilizedfunctionalized microparticle arrays prepared by this process may be usedfor a variety of bioassays, e.g., ligand receptor binding assays.

In one example, thermal hydrogels are formed using azodiisobutyramidinedihydrochloride as a thermal initiator at a low concentration ensuringthat the overall ionic strength of the polymerization mixture falls inthe range of 0.1 mM to 11.0 mM. The initiator used for the UVpolymerization is Irgacure 29590(2-Hydroxy-4′-hydroxyethoxy-2-methylpropiophenone, Ciba Geigy,Tarrytown, N.Y.). The initiator is added to the monomer to give a 1.5%by weight solution.

In certain embodiments, the particle arrays may be immobilized bymechanical means. For example, an array of microwells may be produced bystandard semiconductor processing methods in the low impedance regionsof the silicon substrate. The particle arrays may be formed using suchstructures by, e.g., utilizing LEAPS mediated hydrodynamic andponderomotive forces are utilized to transport and accumulate particleson the hole arrays. The A.C. field is then switched off and particlesare trapped into microwells and thus mechanically confined. Excess beadsare removed leaving behind a geometrically ordered random bead array onthe substrate surface.

When the bead array is immobilized before the assay, the array functionsas a two-dimensional affinity matrix which displays receptors or bindingagents (e.g., oligonucleotides, cDNA, aptamers, antibodies or otherproteins) to capture analytes or ligands (DNA, proteins or other smallcognate ligands) from a solution that is brought in contact with thearray. The bead array platform may be used to perform multiplexedmolecular analysis, such as, e.g., genotyping, gene expressionprofiling, profiling of circulation protein levels and multiplexedkinetic studies.

Substrates (e.g., chips) can be placed in one or more enclosedcompartment, permitting samples and reagents to be transported in andout of the compartments through fluidic interconnection. On-chipimmunoassays for cytokines, e.g., interleukin (IL-6) may be performed inthis format. Serum sample and fluorescent labeled secondary antibodiesare introduced to the reaction chamber sequentially and allowed to reactwith beads immobilized on the chip. FIG. 5 illustrates a design of areaction chamber which may be used in the multiplexed assays accordingto the present invention. Reactions can also be performed in an opencompartment format similar to microtiter plates. Reagents may bepipetted on top of the chip by robotic liquid handling equipment, andmultiple samples may be processed simultaneously. Such a formataccommodates standard sample processing and liquid handling for existingmicrotiter plate format and integrates sample processing and arraydetection.

In certain embodiments, the presence of the analyte-binding agentinteractions are associated with changes in the optical signatures ofbeads involved in the interactions and these optical changes detectedand analyzed. The identities of the binding agents involved in theinteractions are determined by detecting the chemically or physicallydistinguishable characteristic associated with those beads. Preferably,chemically distinguishable characteristics include chemical moleculesincluding flurophore dyes, chromophores and other chemical moleculesthat are used for purposes of detection in binding assays.

The detection of the chemically or physically distinguishablecharacteristic and the detecting of the optical signature changesassociated with the binding interactions may be performed while theparticles are assembled in a planar array on a substrate, e.g., bytaking an assay and a decoding image of the array and comparing the two,e.g., comparing of the assay and the decoding image comprises use ofoptical microscopy apparatus including an imaging detector andcomputerized image capture and analysis apparatus. The decoding imagemay be taken to determine the chemically and/or physicallydistinguishable characteristic that uniquely identifies the bindingagent displayed on the bead surface, e.g., determining the identity ofthe binding agents on each particle in the array by the distinguishablecharacteristic. The assay image of the array is taken to detect theoptical signature of the binding agent and the analyte complex. Incertain embodiments, fluorescent tags (fluorophore dyes) may be attachedto the analytes such that when the analytes are bound to the beads, theflourescent intensities change, thus providing changes in the opticalsignatures of the beads. In certain embodiments, the decoding image istaken after the beads are assembled in an array and immobilized andbefore taking the assay image, preferably before contacting the bindingagents on the beads with an analyte. In certain other examples, thebinding interactions occur while the beads are in solution, andassembled into an array afterwards and the decoding and assay images areobtained.

The identity of the binding agent of the binding agent-analyte complexis carried out by comparing the decoding image with the assay image.

In preferred embodiments, images analysis algorithms that are useful inanalyzing the data obtained from the decoding and the assay images.These algorithm may be used to obtain quantitative data for each beadwithin an array. As summarized in FIG. 7, the analysis softwareautomatically locates bead centers using a bright-field image of thearray as a template, groups beads according to type, assignsquantitative intensities to individual beads, rejects “blemishes” suchas those produced by “matrix” materials of irregular shape in serumsamples, analyzes background intensity statistics and evaluates thebackground-corrected mean intensities for all bead types along with thecorresponding variances.

The methods of the present invention may be used for determining theassociation and the dissociation constants e.g., by introducing theanalyte in a time-dependent manner and analyzing the binding as afunction of time, or by washing away the bound analyte in atime-dependent manner and also analyzing the binding as a function oftime.

The methods of the present invention may be used for determining theaffinity constants of analyte-binding agent interactions, fordetermining the number of analyte-binding agent complexes formed

The present invention also provides methods for determining theconcentration of an analyte in a biological sample.

The methods of the present invention may also be used to determiningelements of a co-affinity matrix of a given analyte against a panel ofbinding agents. In one example, the extent of the interaction betweenthe analyte and the binding agents in a panel in competitive,multiconstituent equilibrium reaction may be determined. Determinationof co-affinity constants provides useful applications, as describedbelow.

The successful rate of transplantation for several types of organsdirectly relates to compatibility of Human Leukocyte Antigen (HLA)between donor and recipient. Serological testing of the recipients forthe Panel Reactive Antibodies (PRA) is one of the crucial steps to avoidpossible rejections. Cross-reaction in PRA testing is a very commonphenomenon due to similarity of some HLA antigen structures and thenature of development of these antibodies. In fact, HLA antigens can beorganized into groups based on apparent serological cross-reactivitybetween the groups. These groups are termed Cross-Reactive-Groups(CREGs). In current clinical setting, antibodies from a patient aretested against different antigens in individual reactions. Although areactive pattern of the antibodies can be generated combining theresults from different reactions, the competitive nature of interactionsbetween different antibodies and antigens is not reflected in such apattern. In other cases, several antigens are mixed together for abinding assay. Lack of identification of each antigen in the systemprevents generation of a binding profile. The result is only theaveraged signal from several antigens. In the bead array system, a panelof different antigens is presented to the antibody analytes in acompetitive binding environment, and each antigen can be identifiedthrough its association with different types of beads. Thus, bindingintensity on each antigen in the competitive reactions can be extractedin a single assay. This co-affinity matrix system will provide bindingprofiles for the CREGs and greatly advance the understanding of thenature of the reaction and improve the accuracy for the related clinicaldecisions. For example, a N-antibody and M-antigen system provides amatrix of N×M of possible reactions. It is possible to determine K-nm,the affinity constant governing the interaction between the nth antibodyagainst the mth antigen, where m=1, 2, . . . M, and n=1, 2, . . . N. Forapplications where absolute co-affinity-constants are not needed,binding profile will be generated for various antibodies in accordancewith the methods of the present invention and results from a patientsample can be matched to these profiles or combination of theseprofiles.

Co-affinity matrix may also be used to characterize the analyte. Forexample, combination of the coefficients of the co-affinity matrix andknown concentrations of analyte and binding agents participating in theformation of analyte-binding agent complexes serves to define acompetitive binding interaction descriptor, e.g., The molecularinteraction parameter,

${P_{n}( R_{m} )} = \frac{K_{mn}\lbrack L_{n} \rbrack}{\sum\limits_{j}\; {K_{mj}\lbrack L_{j} \rbrack}}$

provides a characterization of the molecular interaction between abinding agent, R_(m), and an analyte, L_(n), in the presence of analytes{L_(j); 1≦j≦N}, all of which exhibit a finite affinity, K_(mj), for thatbinding agent. That is, P_(a), 0≧P_(n)≧1, represents a normalizedspecificity of binding agent R_(m) for analyte L_(n) in amulticonstitutent competitive reaction and serves as a robustcharacterization of that binding agent based on co-affinities displayedin a multiconstituent competitive reaction. See also P. H. von Hippel etal., Proc. Natl. Acad. Sci. USA 83, 1603 (1986), incorporated herein byreference.

The pattern of binding interaction of a analyte against a panel ofbinding agents may be used to characterize the analyte and compare itwith other molecules. In addition, by generating the co-affinity matrixof a analyte using a reference panel of binding agents, such affinitymay be used to determine if a sample later introduced to the panel ofbinding agents contains an impurity by observing the deviation in thebinding pattern.

The present invention also provides use of superparamagnetic particles(“magnetic particles”) as described in U.S. Pat. No. 5,759,820 andEuropean Patent No. 83901406.5 (Sintef), which may then be used inintegrated the sample preparation step with the assay step involvingencoded bead arrays. Both of these references are incorporated herein byreference.

Superparamagnetic particles may be encoded with a chemically orphysically distinguishable characteristic (e.g., flourescent tag) andused performing bioassays of the present invention. In certainembodiments, the particles are assembled using LEAPS, as withnon-magnetic encoded beads. The encoded also be used in arraygeneration, and assayed. The present invention also includes theformation of a planar array of encoded and functionalizedsuperparamagnetic particles on a substrate by application of magneticfield to said particles.

Several methods for the synthesis of monodisperse superparamagneticmicrospheres are known in the art. G. Helgesen et al., Phys. Rev. Lett.61, 1736 (1988), for example, disclosed a method which utilizes porousand highly cross-linked polystyrene core particles whose interiorsurfaces are first nitrated, following which iron oxides areprecipitated throughout the particle to produce a paramagnetic core.Following completion of this step, the particles are coated withfunctional polymers to provide a reactive shell. U.S. Pat. No. 5,395,688to Wang et al. describes a process for producing magnetically responsivefluorescent polymer particles composed of a fluorescent polymer coreparticle that is evenly coated with a layer of magnetically responsivemetal oxide. The method utilizes preformed fluorescent polymeric coreparticles which are mixed with an emulsion of styrene and magnetic metaloxide in water and polymerized. A two step reactive process such as thissuffers from the drawback of possible inhibition of polymerization bythe fluorescent dye or conversely bleaching of the fluorescence by theshell polymerization process.

The method also provides a novel process for making color encodedmagnetic beads, a simple and flexible one-step process to introduce intopreformed polymeric microparticles a well controlled amount of magneticnanoparticles, prepared in accordance with the procedure describedbelow, along with well controlled quantities of one or more fluorescentdyes. In an embodiment of the present invention, the quantity of themagnetic nanoparticles is controlled to produce magnetic particles thatform an array on a substrate upon application of magnetic field to saidparticles. This process involves swelling the polymer particles in anorganic solvent containing dyes and magnetic nanoparticles and thereforeapplies to any polymer particle which can be subjected to standardswelling procedures such as those disclosed in the prior art offluorescent staining of microparticles. Unlike encoding methods in whichthe magnetic material and the fluorescent dyes are each located todifferent areas of the (core/shell) of the magnetic particle, uniformswelling of particles ensures the distribution of magnetic particlesthroughout the interior volume. This process also permits thequantitative control of the nanoparticle as well as dye content over awide range, thereby permitting the tailoring of the particles' magneticsusceptibility as well as fluorescence intensities. An additional methodof the present invention to control the magnetic properties of the hostparticles, other than to control loading, is to tune the size of themagnetic nanoparticles by adjusting the water content of the micellarsynthesis reaction (see below).

Physical or chemical coupling of biomolecules possible on the particlesurface utilizing preexisting functional groups. Leaching out ofmagnetic nanoparticles is readily eliminated by growing a furtherpolymeric shell on the particle.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes and are notto be construed as limiting this invention in any manner.

EXAMPLES Example 1 Optically Programmable Array Formation

As illustrated in FIG. 9, LEAPS serves to simultaneously assemblemultiple random encoded subarrays and to “drag-and-drop” these subarraysinto separate, but proximate locations on the chip within a common,enclosed liquid environment. Two sets of beads (2.8 μm Oligo-(dT)₂₅,Dynal, Oslo, Norway), dispensed from separate reservoirs A and B, weresimultaneously assembled into distinct subarrays within the same fluid;sub-arrays were then simultaneously placed into desired destinations asdirected by spatially varying illumination profiles which were generatedand projected onto the substrate by a PC-programmable illuminationpattern generator (described in U.S. Ser. No. 09/397,793, filed Sep. 17,1999, which is incorporated herein by reference in its entirety). Thisdrag-and-drop operation reduced the separation between the twosub-arrays from approximately 250 μm to 20 μm. Beads were moved at 5V_(pp) at a frequency of 2 kHz; total power projected onto the substratesurface was 5 mW. The combination of chemical and spatial encodingpermits a given set of chemical bead markers to be used multiple timesand reduces the demands placed on either encoding dimension whilefacilitating the realization of large coding capacities.

Example 2 Array Formation on Patterned Surface

Illustrated in FIG. 10 is an array of encoded beads assembled on apatterned silicon chip using an AC voltage of 1-2 V_(pp) and a frequencyof 100-150 Hz, applied across a 100 μm electrode gap filled with anaqueous bead suspension; a thermal oxide (˜1000 Å) on the substrate waspatterned by etching the oxide to a thickness of 50-100 Å in a set ofsquare features (˜30×30 dm²) on 130 μm centers; arrays of similar layoutalso can be produced in response to suitable illumination patterns. Eachsub-array shown here contains approximately 80 beads coupled withanti-cytokine monoclonal antibodies. Carboxylate-modified-polystyrenebeads of 5.5 μm diameter (Bangs Laboratory, Fishers, Ind.) were stainedwith a combination of two types of fluorescent dyes and were thenfunctionalized with anti-cytokine-mAb. The assembly process ensurescollection of all beads at the substrate surface. Bead encoding was asfollows: IL-2 (Bright Red); IL-4 (Dim Red); IL-6 (Bright Green); M-CSF(Dim Green) and TNF-α (Yellow).

Example 3 Formation of Arrays of Magnetic Particles

Colloidal particles exhibiting a finite diamagnetic susceptibility, whendisposed on a planar substrate can be assembled into ordered arrays inresponse to increasing magnetic fields. Commercially availablesuperparamagnetic particles (Dynal, Oslo, NO), dispersed from a fluidsuspension onto the planar surface of the lower of two parallel boundingsurfaces of a fluid cell (“sandwich” geometry), when exposed to ahomogeneous axial magnetic field (oriented normal to the substrateplane), will form ordered assemblies. As a function of increasingmagnetic field strength, and for given diamagnetic susceptibility of theparticles as controlled by the manufacturing process known to the art,ordered planar assemblies and linear strings of beads oriented normal tothe substrate can be formed. Permanent magnets can be designed so as toproduce the field strength required to realize the desired configurationof the assembly. Requisite magnetic field configurations can be producedby an electromagnet in solenoid or Helmholtz configuration known to theart; the substrate can be introduced into the magnet bore or can beplaced in immediate proximity to the coil(s) outside of the bore so asto ensure the orientation of the field substantially normal to thesubstrate plane. Spatially modulated magnetic fields can be produced bypatterning the substrate with permalloy using methods known to the art.

Example 4 Formation of Random Bead Assemblies

Aliquots of solution containing suspended beads were placed onto severaldistinct positions on a planer substrate of silicon capped with a thinsilicon oxide layer (other substrates may be used here). Beads wereallowed to settle under gravity to form random assemblies. To delineatediscrete positions on the substrate, one of the following two methodswere used. According to the first method, a silicon gasket (of 250 umthickness), displaying a grid of multiple round holes of 1 mm or 2 mmdiameter (Grace Bio-labs, Bend, Oreg.) is placed on the hydrophillicsurface to define microwells (of 0.25 to 0.5 ul volume) for multiplediscrete samples of bead suspension. According to the second method,small aliquots of fluid containing beads (0.2 ul to 0.5 ul in volume)are directly placed onto a hydrophillic surface in one or moredesignated areas so as to ensure formation of discrete droplets; spacersare not needed in this case. As solvent evaporates (at room temperatureor, for rapid drying, at elevated temperature (about 6° C.), beads areleft in random positions on the substrate. DNA polymorphism reactionshave been tested in assemblies formed in both manners. Optionally, beadssettling under gravity may be immobilized by chemical capture layersprovided on the substrate. An application of random bead assemblies todetermine affinity constants in a multiplexed format is described inExample 6.

Example 5 An Automated Chip-Scale Array Manufacturing Process

As illustrated in FIG. 11, the process involves liquid handling andpipetting of beads onto chips mounted in single-chip cartridges ormulti-chip cartridges. Bead arrays are formed using methods such asthose in Examples 1, 2 or 3, followed by array immobilization anddecoding. The resulting decoding images are stored for later use alongwith an optional chip ID (“bar code”).

Example 6 Determination of Affinity Constants by Post-Assay Analysis ofBead Assemblies

Quantitative binding curves for the cytokines TNF-α and IL-6. Bindingcurves were generated by performing sandwich immunoassays usingchemically encoded beads in suspension, said suspensions being confinedto one or more reaction compartments delineated on-chip, or in one ormore reaction compartments off chip. By completing the reaction withbeads maintained in suspension, assay kinetics similar to homogeneousassays can be attained. Following completion of the binding reaction,beads were assembled on chip to permit multiplexed quantitative imageanalysis. Random assemblies prepared according to Example 4 or orderedbead arrays prepared according to Example 1 or 2 may be used. Anadvantage of ordered, dense assemblies produced by the methods ofExamples 1 or 2 is the higher spatial density and higher assaythroughput attained by processing a greater number of beads.

As an illustration, FIG. 12 displays quantitative binding curves forTNF-α and IL-6, obtained from randomly dispersed beads. Acommercial-grade 8-bit video-CCD camera (Cohu, San Diego, Calif.) wasused in a mode permitting multi-frame integration. The range ofconcentrations of antigen used in the two assays was 700 μM to 50 μM forTNF-α and 2 pM to 50 pM for IL-6. At each concentration, the number ofmolecules bound per bead was estimated by comparison with calibrationbeads coated with known quantities of Cy5.5-labeled BSA per bead;requisite adjustments were made to account for differences influorescence quantum efficiency between labeled secondary antibodies andBSA.

This format of analysis permits the determination of the affinityconstant, K_(A)=[LR]/([R₀−LR][L]), where, in accordance with the law ofmass action, [LR] denotes the number of receptor-ligand pairs perbeadand [L] denotes the solution concentration of ligand. By specifying thenumber of beads per ml, n_(B), and specifying a value for [R₀] in termsof the number of receptors per bead, theoretical binding curves,computed for given K_(A), are compared to a plot of the number of boundmolecules per bead as a function of bulk ligand concentration. Theabsolute number of ligands bound per bead may be determined for givenbulk concentration by measuring the mean fluorescence intensity per beadand referencing this to the fluorescence intensity recorded fromcalibration beads included in the array.

The estimated number of molecules bound per bead is compared totheoretical binding curves derived from the law of mass action. Thethree curves shown correspond to values of the affinity constant, K_(A),of 10¹¹/molar, 10¹⁰/molar and 10⁹/molar, respectively. The initialnumber of antibodies per bead, R₀, equals 2×10⁵/bead and n_(B)=10⁵/ml.Each data point represents the average of three replicates, with anassay-to-assay variation of <45%. Setting the assay sensitivity tocorrespond to that level of fluorescence which yields a signal-to-noiselevel of unity in the assay images, the sensitivity of the cytokineassays characterized in FIG. 12 is set at ˜2,000 bound ligands/bead,corresponding to respective detected concentrations of 700 μM for TNF-αand 2 μM for IL-6.

While commercial ELISA kits use enzymatic amplification to enhancesensitivity, at the expense of additional complexity relating to assayconditions and controls, our bead array assay format, even withoutenzymatic amplification, our on-chip assay format permits monitoring ofcytokines at circulating levels (Normal TNF-α level in serum is 50-280μM and normal IL-6 level in serum is 0-750 μM.www.apbiotech.com/technical/technical_index.html), providing a dynamicrange which approaches that of standard, i.e. amplified single-analyteELISA assays (Assay kits of R & D Systems and Amersham (not the recentHigh-Sensitivity assays). Further improvements at hardware and softwarelevels are possible.

Example 7 Genotyping by Polymorphism Analysis

To illustrate the application of the present invention to theimplementation of genotyping, FIG. 13 shows the design of the assay inwhich five pairs of 20-mer binding agents corresponding to fourpolymorphic regions of a gene were coupled to color-encoded beads. Thepairs of binding agents α1, α2 and β1, β2 each display a singlenucleotide difference in their respective sequences; the pair δ3, δ4displays a difference of three nucleotides, the binding agents in theset γ1, γ3, γ3, γ4 display small insertions and deletions. The tenbinding agents were are divided into two subgroups of five which wereincorporated into two subarrays. In this example, there are severalhundred beads for each type. Following bead immobilization, an on-chiphybridization reaction was performed in TMAC buffer (2.25 Mtetramethylammonium chloride, 37 mM Tris pH 8.0, 3 mM EDTA pH 8.0, and0.15% SDS) at 55° C. for 30 min. The analyte is a 254-base PCR fragmentproduced from a patient sample and fluorescently labeled at the 5′-primeend with BODIPY 630/650 (Molecular Probes, Eugene, Oreg.). Imageacquisition was performed after replacing the assay buffer with freshTMAC buffer.

FIG. 14 shows decoding and assay images for one subarray. Each beadshown in the assay image obtained after hybridization is analyzed todetermine fluorescence intensity and bead type; as with the cytokineassay, the latter operation compares assay and decoding images using atemplate matching algorithm. FIG. 15 displays the resulting intensityhistograms for each bead type: in these histogram plots, the horizontalaxis refers to relative signal intensity from 0 to 1 and the verticalaxises refer to bead numbers. The histograms show that most of the beadsdisplaying probe α1 bind no analyte while most of the beads displayingprobe α2 exhibit significant binding; the mean signal level of α2-beadsexceeds that of α2-beads by a factor of ˜3.2, indicating that analytecontains DNA sequences complementary to α2 but not α1. For the patientsample presented here, the histogram indicates a genotype of the analyteDNA characterized by complementarity to binding agents α2, β2, γ3, γ4and δ4 in the polymorphic region of the gene.

Example 8 Gene Expression Analysis cDNA Fragments

The method of the present invention has been used to fabricate arrayscomposed of beads displaying oligonucleotides as well as DNA fragments(e.g., up to ˜1,000 bases in length). Strands were biotinylated atmultiple positions by nick-translation and were attached tostreptavidin-functionalized beads (M-280, Dynal, Oslo, NO). Arrays wereformed using an AC voltage of 800 Hz at 10V_(pp).

Example 9 Looped Probe Design for Universal Labeling

A looped probe design in FIG. 16 takes advantage of fluorescence energytransfer to obviate the need for labeled target. As with the molecularbeacon design (S. Tyagi, D. P. Bratu. F. R. Kramer, Nature Biotech. 16,49-53 (1998)), the probe in FIG. 16 assumes two different states offluorescence in the closed loop and open loop configurations, but incontrast to the molecular beacon contains a portion of its binding motifwithin the stem structure to permit molecular control of stringency incompetitive hybridization assays.

Example 10 Quantitative Multiplexed Profiling of Cytokine Expression

FIG. 17 displays a pair of assay and decoding images recorded from asingle random array in a multiplexed sandwich immunoassay. An arraycontaining five distinct types of beads, each displaying a monoclonalanti-cytokine antibody (mAb), was exposed to a sample solution (such asserum) containing two cytokine antigens (Ag). Subsequent addition ofCy5.5-labeled secondary antibodies (pAb*) results in the formation ofternary complexes, mAb-Ag-pAb*. The on-chip immunoassay was performed byadding 300 μl of sample with 7 nM cytokines in assay buffer (20 mM NaPipH 7.4, 150 mM NaCl*, 10 mg/ml BSA) to the bead array immobilized on thechip, and allowing the reaction to proceed at 37° C. for one hour. Thebuffer was replaced by adding 12 nM solution of labeled secondaryantibodies in assay buffer. After one hour of incubation at 37° C.,fresh buffer was added on top of the chip and image acquisition wasperformed. Antibodies and antigens used in the assays were obtained fromR&D Systems (Minneapois, Minn.); the secondary antibody was labeled withCy5.5 using a standard kit (Amersham Pharmacia Biotech, Piscataway,N.J.).

The decoding image FIG. 17B shows five types of beads in a false-colordisplay with the same encoding pattern as that of FIG. 10. All beads areof the same size (5.5 μm diameter); the apparent difference in the sizeof beads of different types in the decoding image is an artifactreflecting different internal bead staining levels and “blooming” duringCCD recording of the decoding image. Comparison (using the imageanalysis methods disclosed herein) of the decoding image with the assayimage in FIG. 14A reveals that active beads, of yellow and bright greentypes, captured TNF-α: and IL-6, respectively. This assay protocol hasbeen extended to the following set of twelve cytokines: IL-1αIL-1β,IL-2, IL-4, IL-6, TGF-β1, IL-12, EGF, GM-CSF, M-CSF, MCP-1 and TNF-α.The on-chip immunoassay requires no additional washing other thanchanging reagent solutions between assay steps. Comparison between assayand decoding images shows that two different cytokines were present inthe sample, namely IL-6 and TNF-α. The pre-formed arrays described inthis example also permit the determination of affinity constants in amanner analogous to the analysis described in Example 6.

Example 11 Aptamers for Protein Profiling

Aptamers may be selected from large combinatorial libraries for theirhigh binding affinities to serum proteins (L. Gold, B. Polisky, O.Uhlenbeck, M. Yarus, Annu. Rev. Biochem. 64: 763-797. (1995)). Randomencoded arrays of aptamer-functionalized beads would serve to monitorlevels of serum proteins; correlations in binding patterns on the array(see also Example 10) may serve as a phenotype of disease states.

Example 12 Mixed DNA Protein Arrays

Of significant interest to genomic functional analysis is the fact thatthe method of the present invention accommodates protein and DNA arrayswithout change in array manufacturing methodology. Specifically, mixedarrays composed of beads displaying DNA and corresponding proteins canbe used to analyze the gene and gene product within the same fluidsample.

This has been demonstrated for a combination of immunoassay and DNAhybridization. For example, a mixed array composed of beadsfunctionalized with anti-cytokine monoclonal antibodies (mAb) and witholigonucleotides was produced. Two sequential assays were performed onthis single chip. First, an immunoassay was performed in accordance withthe protocol described in Example 10. Following completion of theon-chip immunoassay, image is acquired and the DNA analyte was added tothe hybridization buffer (2×SSC, 1×Denhardt's) at a final concentrationof 20 nM and allowed to react at 37° C. for 1 hr. Fresh hybridizationbuffer was added to the chip and image acquisition was performed torecord of the additional hybridization assay.

Example 13 Affinity Fingerprinting

The analysis of receptor-ligand interactions relevant to prior artmethods assumes ideal specificity. That is, only the ideal situation isconsidered of a single ligand present in solution reacting with itsmatching receptor and vice versa. However, in most multiple assaysystems, a considerable level of cross-reactivity may exist. That is,any single ligand may associate with several receptors, while any singletype of receptor may have a finite affinity towards more than oneligand.

The present invention includes a model that is developed to analyzemultiplexed READ assays for such a system under the followingassumptions: each of these reversible reactions is characterized by itsown affinity constant; no reaction occurs between the bulk species;there is no interaction between the complexes formed on the surface.These assumptions can be relaxed, at the expense of increasing thecomplexity of modeling, by accounting for reactions in the bulk andbetween the surface species. The standard reaction-diffusion equationfor single receptor-ligand pair formation [R. W. Glaser, Anal. Biochem.213, 152-161 (1993)], is generalized to allow for multiple reactions ateach bead surface:

$\begin{matrix}{\frac{\partial\lbrack {L_{i} \cdot R_{j}} \rbrack}{\partial t} = {\quad{{{{k_{{on},{ij}}\lbrack L_{i} \rbrack}\begin{pmatrix}{\lbrack R_{j,0} \rbrack -} \\{\sum\limits_{n,m}\; \lbrack {L_{m} \cdot R_{n}} \rbrack}\end{pmatrix}} - {{k_{{off},{ij}}\lbrack {L_{i} \cdot R_{j}} \rbrack}{\forall i}}},j,{L_{i} \equiv {L_{i}( {t,x,0} )}}}}} & (2)\end{matrix}$

The first term on the right of Eq. (1) describes the association ofligands and receptors into complexes and involves of concentration offree sites on the surface. The second term describes the disassociationof complexes by way of release of ligands, thereby freeing up receptorsites for further reaction. Since a maximum of (i×j) bimolecularcomplexes can form, there could be as many boundary conditions-generatedfrom the above equation. For the equilibrium case, the left hand-side ofEq. (1) is set to zero, and the matrix of coaffinities,[K_(ij)]=k_(on,ij)/k_(off,ij), can then be defined to accommodatecross-reactivities between multiple species in the bulk and on thesurface. In a batch reactor under equilibrium conditions, we may solvethe system of differential equations to obtain the number of moleculesof each ligand bound on beads of each type.

L₁ Ligand concentration  10 pM L₂ Ligand concentration 100 pM R₀₁Initial receptor concentration 1 × 10⁴/bead R₀₂ Initial receptorconcentration 1 × 10⁴/bead n_(B1) Bead number density 1 × 10⁴/ml n_(B2)Bead number density 1 × 10⁴/ml [K] Coaffinity matrix [1 × 10¹¹ 1 × 10⁹ 1× 10⁸ 1 × 10¹¹] l/mole

As an illustrative example, the ligand distribution has been calculated(from the model in Eq (1)) for a reference set of two ligands and twotypes of receptors immobilized on two different sets of beads. Thecoaffinity matrix is assumed known for each ligand-receptor combinationin the reference set; to investigate the detection of a third ligand, itis assumed here that diagonal elements of the 2×2 matrix, [K_(ij)], arelarge compared to off-diagonal elements. The presence of a third ligandin the reactor alongside the two original ligands perturbs theequilibria between the various complexes and the reactants in thereference system, and for ligand molecules tagged with fluorescentlabels, the intensity observed from the perturbed system differs fromthat observed in the reference case.

FIG. 18A shows the reference case in which the concentrations andcoaffinity matrix were set to the values shown in the accompanyingtable; the bead intensity was defined on a linear scale of 0-255, thelatter representing the intensity of the brightest beads. FIG. 18A showsthe contribution of each ligand to the bead intensity. Due to the lowerconcentration of L₁, the intensity of Bead 1 is less compared to Bead 2,cross-reactivities are essentially undetectable.

Next, the system was perturbed with a third ligand, taking theconcentration L₃ to be 1 pM and assuming that the new ligand hasconsiderable amount of cross-reactivity with each of the receptors;K_(3,1)=1×10¹¹/M, K_(3,2)=1×10¹⁰/M. Calculation of the fluorescentintensity of each bead in the presence of the third ligand yields thepattern in FIG. 18A which reveals an increase in the intensity of Bead 1due to the third ligand, while leaving the intensity of Bead 2unaffected due to the higher concentration of L₂ in the system and thelower affinity of L₃ to R₂. Thus, L₃ may be detected under the conditionthat it has a relatively high affinity to one of the receptors and is insignificant amount compared to the competing ligand.

The evaluation of the coaffinity matrix (and comparison with theoreticalmodeling as disclosed herein) under conditions in which a mixture ofligands is permitted to interact with a multiplicity of receptorsarranged in a random encoded bead array format provides a methodology toestablish a characteristic feature set of cross-correlations in themutual competitive binding affinities of multiple ligands and receptors.These co-affinities provide a robust means to characterizereceptor-ligand binding equilibria by their affinity fingerprintingpatterns. Deviations from well-defined reference cases also permitdetection of “perturbing” ligands in solutions.

Example 14 Multiplexed Analysis of Reaction Kinetics

As illustrated in the foregoing examples, extensive washing generally isnot required to discriminate beads from a background of solutionfluorescence. Consequently, assay image sequences may be recorded in ahomogeneous assay format to document the evolution of a binding reactionand to determine kinetic data for each of the binding reactionsoccurring.

Homogeneous binding assays may be performed in-simple “sandwich” fluidiccartridges permitting optical microscopic imaging of the bead array andpermitting the introduction of an analyte solution into a chambercontaining a random encoded array of beads. More generally, the arrayalso may be exposed to an analyte or other reaction mixtures underconditions of controlled injection of fluid aliquots or continuous flowof reactants or buffer. Using theoretical modeling, optimal combinationsof relevant performance control parameters of this bead array reactormay be identified to minimize the time to equilibration or to maximizethe portion of analyte captured by the array [K. Podual and M. Seul, TMKP-99/02]. Flow rate can be controlled by any of a number of availablepumping mechanisms [M. Freemantle, C&EN, 77: 27-36].

TABLE List of parameters used in simulations (FIG. 18) Parameter, unitsValue Initial Receptor Coverage c_(R,0), moles/m² 8 × 10⁻⁹ Vol FlowRate, Q, μl/s 1.0 Diffusivity, D, cm²/s 1 × 10⁻⁷ ON-Rate, k_(on), /(M s)1 × 10⁵  Affinity Constant, K_(A), /M 1 × 10¹¹ “Sandwich” Reactor GapSize H, mm 0.1 Reactor Length, L, mm 10 Reactor Width, W, mm 10

The analysis of image sequences permits kinetic data to be generatedfrom which ON-rates and OFF-rates are determined with the aid of atheoretical model of the reaction-diffusion kinetics of the typeillustrated in the foregoing example in FIG. 19. FIG. 19A displaysstages in an adsorption-desorption cycle involving solution-borneanalytes and a bead array immobilized at the bottom of a “sandwich”reaction chamber. The first panel depicts the initiation of theadsorption process; the second panel depicts the state of the reactorclose to equilibrium when most of the beads have reached equilibrium;the last panel depicts the state of the reactor under the desorptioncycle in which ligand-free fluid is injected and adsorbed moleculesdesorb from the bead surface. FIG. 19B displays theadsorption-desorption kinetics of a single receptor-single ligand systemobtained by numerical solution of a reaction-diffusion system for asingle type of receptor-ligand reaction; two cases of differentconcentrations of ligand are shown. Parameters used in the simulationare listed in the accompanying Table.

In contrast to prior art methods [D. G. Myszka, Curr. Opin. Biotechnol.8: 50-57.], the present method relies on imaging and permitsmultiplexing. In addition, generalized models of the type introduced inExample 6 permit the analysis of complex binding kinetics for multiplesimultaneous receptor-ligand interactions even in the presence ofcross-reactions between multiple ligands and receptors.

The ability to monitor reaction kinetics in an array format will enableseveral approaches to enhancing the specificity of receptor-ligand orbinding agent-analyte interactions in complex mixtures. For example,temperature programming may be invoked to enhance the specificity of DNAhybridization reactions. Similarly, the stringency of conditions appliedto a hybridization reaction may be varied while the array response isbeing monitored; for example, hybridization may be conducted in ahybridization buffer under conditions leading to excess “non-specific”binding; specificity is enhanced by switching to a wash buffer ofincreasing stringency while monitoring the array response.

Example 15 Multi-Step Assay Sequences Using Encoded Arrays of MagneticParticles

Methods and apparatus using biochemically functionalizedsuper-paramagnetic particles for sample preparation in molecular andcellular biology and for a variety of enzyme-catalyzed on-bead reactionshave been described [“Biomagnetic Techniques in Molecular Biology”,Technical Handbook, 3^(rd) Edition, 1998, Dynal, Oslo, NO]. Thesebead-based methods can be combined with the Random Encoded ArrayDetection format of the present invention to implement multi-stepon-chip assay manipulations.

For example, FIG. 20 illustrates the integration of a sequence of stepsin a miniaturized format for multiplexed genotyping using a single chipwith multiple compartments. First, cells are captured from a patientsample by affinity selection using functionalized magnetic beads, cellsare lysed electrically or chemically in a first compartment, and genomicDNA is captured to the surface of a multiplicity of magnetic beads bynon-specific binding; next, beads are collected by magnetic force into asecond compartment which is in fluidic contact with the firstcompartment, within which the beads and DNA are washed with desiredbuffers; next, beads are further transferred to a location where PCR isperformed using bead-coupled DNA as a template; multiple PCR strategiesknown in the art are available for this step [F. Fellmann, et. al.,Biotechniques, 21:766-770]; next, PCR products released into arecaptured by hybridization to a pre-assembled random encoded arraydisplaying binding agents that are specific to different polymorphismstargeted by the PCR amplification.

The use of encoded magnetic particles in conjunction with the opticalprogrammability of LEAPS confers the ability to form reversiblyimmobilized arrays and to conduct programmable multi-step assaysequences under conditions in which beads are used in suspension whenthis is most favorable, for example to enhance reaction kinetics, andarrays are formed in real-time when this is most favorable, for exampleto provide a highly parallel format for imaging detection and assayread-out.

For example, as illustrated in FIG. 21, the following sequence of stepscould be integrated in a miniaturized format for the formation of a cDNAbead array. First, a pool of encoded magnetic beads, each bead typedisplaying a gene-specific probe, is introduced to an mRNA pool, andmRNA molecules are hybridized to their corresponding beads; next,on-bead reverse transcription (RT) is performed using bead-attached mRNAas template [E. Horenes, L. Korsnes, US 005759820]; next mRNA isreleased from the beads; next beads are directed to the surface of acustom-designed chip and a cDNA bead array is formed using LEAPS. Suchan array could serve to display binding agents in a gene profilingexperiment using another set of mRNA as the target. Alternatively, thecDNA array could be analyzed for its own expression by applying a poolof labeled DNA binding agents to profile the genes of interest withinthe array.

Example 16 Synthesis of super-paramagnetic iron oxide γ-Fe₂O₃(maghemite) particles

The synthesis was carried out in reversed micellar solutions composed ofthe anionic surfactant, bis(2-ethylhexyl)sodium sulfosuccinate (AOT) andisooctane (Kommareddi et al., Chem. Mater. 1996, 8, 801-809)obtainedfrom Aldrich Chemical Co., Milwaukee, Wis. Stock solutions of 0.5M AOTwere used in preparing the reversed micellar solutions containing thereactants FeSO₄ (Sigma Chemical Co., St. Louis, Mo.) and NH₄OH (SigmaChemical Co., St. Louis, Mo.). Specifically, 0.45 ml of 0.9M FeSO₄ wasadded to 5 ml of 0.5M AOT in isooctane, separately 0.45 ml of NH₄OH wasadded to 5 ml of 0.5M AOT in isooctane. The reaction was initiated byadding the NH₄OH reversed micellar solution to the FeSO₄ reversedmicellar solution under vigorous stirring. The reaction was allowed toproceed for 2-3 hrs and then the solvent was evaporated at ˜40° C. toobtain a dry surfactant iron oxide composite.

This composite was re-dispersed in the organic solvent of choice to givea deep red colored transparent solution.

Example 17 Synthesis of Fluorescently Colored and Magnetic Polymer BeadComposites

A stock solution of hydrophobic fluorescent dye and the iron oxideparticles was made by re-dispersing the dried magnetic composite and thedye in the solvent of choice, for example a CHCl₃ (Aldrich Chemical Co.,Milwaukee, Wis.) or CH₂Cl₂CH₃OH mixture (70/30 (v/v)) (Aldrich ChemicalCo., Milwaukee, Wis.). A predetermined amount of polymer beads waswashed thoroughly in methanol (3×) and then evaporated dry. Simultaneousincorporation of the fluorescent dye and the iron oxide nanoparticle wasachieved by swelling the beads in organic solvent/nanoparticle/dyemixture. The swelling process was completed within 1 hr. Following thisthe polymer beads were separated by centrifugation and washed withmethanol(3×) followed by isooctane(2×) and then methanol(2×) and finallyredispersed in 0.2% SDS-DI water solution.

1-40. (canceled)
 1. A method of real time monitoring of a multiplicityof target-oligonucleotide probe binding reactions carried out in thesame reaction vessel, where detection of reaction results is performedby imaging signals which indicate binding of said targets tooligonucleotide probes, said oligonucleotide probes being bound toencoded carriers, wherein different types of oligonucleotide probes arebound to differently-encoded carriers and signals associated withdifferently-encoded carriers can be discriminated, comprising:conjugating said carriers with oligonucleotide probes which include twocomplementary subsequences, separated by a third subsequence, andwherein a first complementary subsequence has a greater number ofnucleotides which are complementary to the target subsequence than tothe other complementary subsequence; providing conditions such that saidtwo complementary subsequences are annealed in the absence of target,but wherein the binding of the target subsequence to the firstcomplementary subsequence causes separation of said two complementarysubsequences and generation of a fluorescent signal; forming saidcarriers into a planar array by placing them on a planar surface whichis placed inside the reaction vessel; adding targets, in solution, tothe reaction vessel; imaging said planar array to detect said signals ata first time and at least one other time, to thereby monitor differencein signals between the two times and to thus monitor the relativequantity target-ligand binding at said first and other times; andidentifying the types of ligands binding to targets by decoding thecarriers associated with signals.
 2. The method of claim 1 wherein thesignals are monitored continuously.
 3. The method of claim 1 wherein theimages are recorded.
 4. The method of claim 3 wherein binding of thetarget subsequence is detected by means of detecting fluorescence from apair of fluorescent moieties respectively associated with the first andother complementary subsequence.
 5. The method of claim 4 wherein thebinding of the target subsequence is detected by means of detectingfluorescence from the donor in a donor-acceptor pair of fluorescentmoieties respectively associated with the first and second subsequences(the members of the pair capable of interacting, when the first andsecond subsequences are annealed) by fluorescence energy transfer.